PROJECT SUMMARY The objective of this proposal is to define the critical role that SIRT2 plays in directing the DNA damage response (DDR) through post-translational modification of the acetylome, which places it at the confluence of cellular metabolism, aging, carcinogenesis, and development of tumor cell resistance to anticancer agents. SIRT2 is a sirtuin family deacetylase that sense energy requirements and direct cellular processes to maintain metabolic homeostasis. Significantly, mice deficient in Sirt2 develop breast, liver, and other cancers, implying that SIRT2 is a tumor suppressor. Paradoxically, SIRT2 is overexpressed in many human cancers, including breast malignancies, and furthermore, our data suggest that high SIRT2 expression is associated with poor survival in breast cancer patients treated with adjuvant ionizing radiation (IR) and contributes to resistance to many types of cancer treatments, including IR, PARP inhibitor, and chemotherapy. However, the precise mechanisms by which SIRT2 directs the DDR to govern tumor cell treatment resistance, and moreover, how SIRT2 itself is regulated in the DDR are poorly understood. In the previous funding period, we defined a novel role for SIRT2 in directing the replication stress response (RSR) through the acetylation status of ATRIP and CDK9, and furthermore, showed that somatic SIRT2 mutations impair the activity of SIRT2 in maintaining genome integrity. In this renewal application, we propose to carry this work further by investigating a new role for SIRT2 in directing the repair of DNA double-strand breaks (DSB) through deacetylation of MRE11, identifying the upstream ubiquitination signaling events governing SIRT2 activity, and determining if SIRT2 inhibition is an effective therapeutic strategy for treatment of breast cancers resistant to conventional therapies. In this regard, we have shown that SIRT2 deacetylase activity mediates resistance of cancer cells to IR and PARP inhibitor by facilitating the end resection step in homologous recombination repair. We further identified a novel network of DDR proteins deacetylated by SIRT2 in response to IR, including MRE11. Moreover, our data suggest that SIRT2 activity but not levels is regulated in the DDR via protein monoubiquitination. We hypothesize that SIRT2 responds to upstream ubiquitination signaling to maintain genome integrity and govern breast cancer treatment resistance by directing the DDR through deacetylation of key substrates, including MRE11, which may be exploited to improve breast cancer control. We propose to: 1) Determine the role of SIRT2 in directing MRE11 in the DDR; 2) Dissect the mechanism by which SIRT2 is regulated by monoubiquitination in the DDR; 3) Establish SIRT2 as a new molecular target for breast cancer resistant to DNA damaging agents. Completion of this work will provide a detailed mechanistic understanding of how SIRT2 directs DSB repair to maintain genome integrity and govern tumor cell treatment resistance, define a novel mechanism for regulation of SIRT2 via protein monoubiquitination in the DDR, and establish proof of concept for SIRT2 inhibition as novel therapeutic approach for overcoming breast cancer treatment resistance.